Extracting DNA3/15

Integrated Science 4Name:Per:

Background

DNA is a nucleic acid found in the nucleus of cells that stores and transmits genetic information from one generation of an organism to the next by coding for the production of a cell’s proteins. Bacteria, fungi, plants, animals and all other living organisms on this planet contain nucleic acids. The DNA of eukaryotic cells (all of the above except bacteria) is well protected inside the nucleus - DNA can even be isolated from dead organisms. In order to perform any type of study or analysis on DNA, it is necessary first to get inside the cell and, then, to get inside the nucleus. Once the nucleus is opened the extraction and isolation is possible because DNA is a very stable molecule with distinctive properties. These properties include: a clear, glassy appearance when wet, an ability to stick to itself, solubility in water, and insolubility in an alcohol or salt mixture.

In this lab, we will use a simplified version of the standard technique used in the biotechnology industry to extract DNA from plant and animal cells. In the extraction, an extraction buffer containing detergent and salt will be used. Detergent breaks down cell membranes and nuclei, isolating the DNA. Salt induces the DNA to clump together. To collect the DNA, cold ethanol is effective because DNA is less dense than and insoluable in alcohol. Once extracted, this DNA is available for analysis, cloning, or recombination.

As you work, consider the following focus questions:

  • How is DNA extracted from a eukaryotic cell?
  • What is the relationship between the appearance of precipitated DNA to its known physical properties?
  • How can extracted DNA be used?

Materials

  • Strawberry Extraction: strawberry, Ziploc bag, extraction buffer (detergent, salt, and water), test tube, funnel, stir rod, cheesecloth, 10 ml graduated cylinder, ethanol (alcohol)

Procedure

There are three steps to DNA extraction: A. breaking down the tissue to access the cells, B. breaking open the cell membranes and nuclei of the cells to release the DNA, and C. collecting the DNA.

  1. Breaking Down the Tissue
  1. Take a strawberry and place it in a Ziploc bag. Smash the strawberry for about 2 minutes. You need to completely crush the strawberry.
  1. Breaking Open the Cell Membranes and Nuclei
  1. Add 5 ml of extraction buffer into the bag and smush for another minute. Be careful not to make too many bubbles.
  2. Place a piece of folded cheesecloth into a funnel. Insert the funnel into the test tube.
  3. Pour some of the mixture through the cheesecloth and allow it to filter into the test tube. Allow only about an inch of liquid to filter through into the test tube.
  4. Remove the cheesecloth, throw it away, and then remove the funnel. Clean and dry funnel.
  1. Collecting the DNA
  1. Note the level of the liquid in the test tube. Note the point on the test tube that is double that volume.
  2. SLOWLY and CAREFULLY add 5 ml cold ethanol (alcohol) down the side of the tube to the level of the mark on the test tube.
  3. At this point you will see a snotty, white precipitate at the point where the liquid and the alcohol meet. This is the DNA. DNA cannot dissolve in ethanol, so the molecules will clump together and become visible. The colder the ethanol, the less soluble the DNA will be, and the more visible clumping.
  4. Use a pipet or glass stirring rod to pull out the DNA by spinning it like you’re making cotton candy. You may need to use forceps to help if the DNA slips off. Each fiber contains millions of DNA strands!
  5. Compare your DNA with your partner groups. Then either save or dispose of DNA as indicated by your teacher. Clean and dry all materials.

Analysis and Conclusion

  1. What was the purpose of A.crushing the tissue? What did you observe that indicated this had occurred? Include detailed descriptions of your observations.
  1. What was the purpose of B. the DNA extraction buffer have on the cells? What did you observe that indicated this had occurred? Include detailed descriptions of your observations.
  1. What effect did C. adding the ethanol (alcohol) have on the DNA? What did you observe that indicated this had occurred? Include detailed descriptions of your observations.
  1. In order to study our genes, scientists must extract DNA from human tissue. If you wanted to extract DNA from a living person, which cells would you use and why? What could this technology be used for?